Although N gonorrhoeae is an obligate human pathogen, it has the capacity to infect and cause disease at numerous sites. However, this capacity requires that gonococci (gc) resist antibacterial substances that naturally bathe mucosal surfaces or become available due to inflammation. The emphasis of this project is to understand the mechanisms used by gc to resist antibiotic-like substances that bathe certain mucosal sites. The mtr (multiple transferrable resistance) system is of likely importance in the ability of gc to infect and cause disease at certain mucosal sites because it modulates levels of resistance to structurally diverse hydrophobic agents (HAs), including bile salts, fatty acids, lysosomal proteins and antibiotics. Recent evidence suggests that mtr mediated resistance to HA is due to an energy-dependent afflux process that removes structurally diverse HAs that exert antimicrobial action at the membrane or cytoplasmic levels. Levels of the afflux pump which is encoded by the mtrCDE genes, and resistance to HA are regulated by the mtrR gene. The mtrR gene encodes a 210 amino acid protein that resembles certain DNA-binding proteins that act as transcriptional repressors. Mutations in either the mtrR-coding sequence or a 13 base pair inverted repeat sequence within the mtrR promoter enhance gonococcal resistance to HA and appear to enhance levels of the mtrCDE-encoded afflux pump. The mtrCDE genes are organized as a single transcriptional unit, downstream and divergent from the mtrR gene. The complete amino acid sequence for the mtrC gene was deduced from DNA sequencing studies and it belongs to a family of bacterial proteins (termed membrane-fusion proteins MFP) that participate in energy-dependent removal of diverse antibiotics from either the piroplasm or cytoplasm. While only partial DNA sequences are available for the mtrD and mtrE genes, the available data indicate that their encoded proteins resemble other bacterial proteins that complex with members of the MFP family to form afflux pumps that extend from the cytoplasm to the outer membrane. The experiments outlined in the specific aims of this proposal will address the structure-function relationships of the MtrD and MtrE proteins in mediating afflux of structurally diverse HA, whether the mtr system is constitutively expressed or is inducible by HA and the mechanisms by which MtrR-dependent and independent systems regulate expression of the mtrCDE operon. The results from these studies will advance our knowledge regarding the mechanisms utilized by gc and other pathogens to avoid antimicrobial agents present on mucosal surfaces or antibiotics used in therapy of bacterial diseases. The specific aims are to: 1) Complete the sequencing of the mtrCDE operon and construct null mutations in the mtrDE genes to determine the role of their encoded proteins in mediating HA resistance. 2) Examine uptake and afflux of HAs using isogenic strains differing in the MtrC, MtrD and MtrE proteins and 3) Define the mechanisms by which the mtr operon is regulated.